Time-Resolved Fluorescence Spectroscopy is a powerful tool for biochemistry; it can provide unique insights into the structure and assembly of macromolecular complexes. This year, we mainly studied ultrafast protein solvation, mitochondrial energetics and ultrafast microscopy development. [unreadable] We also continued bending studies of DNA using fluorescent nucleotide analogs that reveal disruptions in DNA shape (e.g., mismatch or hairpins) and translational diffusion (FCS below).[unreadable] We continued and expanded our femtosecond upconversion studies of Trp in proteins and peptides to quantify early "quasistatic self-quenching" processes. We found extremely rapid (10-100ps) decays are important in several proteins (crystallins, thioredoxin, GB1,etc.), as they imply conformers with ultrafast charge transfer. Our earlier study of protein *solvation* on the 330fs-200ps time scale, using proteins such as Monellin, found QSSQ. Others hypothesized waters were desorbing from protein in 20ps, when, in fact, the fast decay process (QSSQ) misled their analysis. We demonstrated (with several other proteins this year) that local quenching is the key mechanism in all but a locally unstructured protein.[unreadable] We contined collaborative studies with LCE into the status of a primary fuel of heart muscle mitochondria- NADH. Our efforts distinguish free and bound populations of NADH by their different fluorescence lifetimes, and we expanded these studies in our new 2-photon fluorescence lifetime microscopy facility (collaboration with Microscopy Core) , obtaining Decay-Associated Images of NADH binding within isolated cardiac myocytes. This year, we added a CARS (Coherent AntiStokes Raman Spectroscopy) capability that allows us to view the nonfluorescent oxidized form of NADH, NAD+.[unreadable] We have used our 2-photon FCS (Fluorescence (Cross) Correlation Spectrometer) with imaging capabilities to study the transport and binding of fluorescent proteins in both stably and transiently transfected cells. Our study of androgen receptor ( binding and cotransport with the Tif2 cofactor in the nucleus)was published. The level of interaction changed with effector drugs . We began examining integrase assembly on HIV-LTR DNA and the integration host factor/HU bending of DNA with the same system. FCCS is a useful tool for quantifying mobility and stoichiometry of dilute proteins either in solution or in a cell. The same system was used to study the interactions of the HIV nef protein with transport proteins (organelle dependent). We also employed FCS to study the mobility of very low levels of C-myc and (separately) Mu transposase.[unreadable] We built (and filed for patents) light collection devices for multiphoton microscopy of tissue whose purpose is to salvage any light that is emitted by the sample but does not enter the pupil of the objective. We showed that deep in rat brain slices, GFP labeled myosin gave >6X brighter signals using our (Total Emission Detection (Morelight ) device. A manuscript is now accepted for publication.[unreadable] We began collaborative simulation of deep multiphoton imaging with NICHD to seek the theoretical limits of such devices, with or without phase compensation array devices.